Stable long-term indigo production by overexpression of dioxygenase genes using a chromosomal integrated cascade expression circuit

In our laboratory we have analyzed different factors to maximize the yield in heterologous protein expression for long-term cultivation, by combination of an efficient cascade expression system and stable integration in the bacterial chromosome. In this work, we have explored this system for the production of indigo dye as a model for biotechnological production, by expressing in Escherichia coli the thnA1A2A3A4 genes from Sphingomonas macrogolitabida strain TFA, which encode the components of a tetralin dioxygenase activity. We compared Ptac, and the Pm-based cascade expression circuit in a multicopy plasmid and stably integrated into the bacterial chromosome. Plasmid-based expression systems resulted in instability of indigo production when serially diluted batch experiments were performed without a selective pressure. This problem was solved by integrating the expression module in the chromosome. Despite the gene dosage reduction, the synergic effect of the cascade expression system produced comparable expression to the dioxygenase activity in the plasmid configuration but could be stably maintained for at least 5 days. Here, we show that the cascade amplification circuit integrated in the chromosome could be an excellent system for tight control and stable production of recombinant products.     

mRNA stability and plasmid copy number effects on gene expression from an inducible promoter system

The effects of mRNA stability and plasmid copy number on gene expression in Escherichia coli were evaluated by constructing multicopy (pMB1-based) and low-copy (F-based) plasmids containing an arabinose-inducible promoter system, the lacZ reporter gene, and mRNA-stabilizing 5' hairpin structures. Product formation and cell growth were evaluated under a number of inducer concentrations. The introduction of a 5' hairpin into the untranslated region of the mRNA resulted in significantly higher gene expression from the multicopy plasmids at low inducer concentrations and increased gene expression from the low-copy plasmids across all inducer concentrations investigated. With high inducer concentrations, expression from high-copy plasmids significantly slowed cell growth, whereas expression from the low-copy plasmids had little effect on growth rate. At inducer concentrations between 1 x 10(-4) and 4 x 10(-4)%, the productivity of low-copy plasmids containing the 5'-hairpin was equal to or greater than that from multicopy plasmids. Together, these two gene expression strategies may find important use in metabolic engineering and heterologous gene expression.     

Design of Streptomyces nogalater LV65 strains with higher synthesis of nogalamicin using regulatory genes

Influence of cloned regulatory genes on biosynthesis of nogalamicin by Streptomyces nogalater LV65 strains has been studied. Gene snorA from the S. nogalater genome was cloned in multicopy replicative plasmid pSOKA and integrative plasmid pR3A. Introduction of these plasmids into the cells of wild type strain of S. nogalater LV65 resulted in higher synthesis of nogalamicin. A similar effect was observed at heterologous expression of gene ppGpp of synthetase relA cloned in S. coelicolor A3(2). Heterologous expression of genes absA2from S. ghanaensis ATCC14672 and lndyR from genome S. globisporus 1912 decreased synthesis of antibiotic. The study results indicate the presence of homologs of these genes in chromosome of S. nogalater, their possible participation in regulation of nogalamicin biosynthesis, and provide us with a possibility for genetic design of the strains with higher synthesis of this antibiotic.     

Saccharomyces cerevisiae mutants with increased mitotic stability of plasmids isolated during long-term culturing of transformants under selective conditions]

Analysis of the plasmid containing clones of transformants of Saccharomyces cerevisiae in the population cultivated under the nonselective conditions has shown their vast heterogeneity in the mitotic stability of the plasmids Yep13 and Yep91. For instance, the clones were obtained with the different types of the hereditary plasmid stabilization: integration with the chromosome and genotype or plasmid mutations increasing the vector copy number. The increased expression level was registered in the mutants for the heterologous genes AmpR of Escherichia coli and HBsAg of hepatitis B. The clones were found with the considerably varying mitotic stability of the plasmids of the modification type variability, the latter expressing the fluctuations of plasmid copy number at the change of cultivation conditions.     

Cloning of the integration and attachment regions of bacteriophage P4

Summary The integration and attachment regions of bacteriophage P4 have been cloned into a multicopy plasmid. This plasmid can integrate into the E. coli chromosome at the same location as the parent phage. Integration increases the stability of the plasmid and allows it to be retained even under conditions in which a non-integrated plasmid would be lost. None of the genes needed for P4 lytic growth is required for integration. The P4 integration and attachment regions have been cloned on separate plasmids. A plasmid that carries the attachment site can integrate into the chromosome only if another plasmid that carries the P4 integration functions is present. A plasmid that carries only this trans-acting integration function cannot integrate. Using deletion mutants of the plasmid, the maximum size of the region needed for integration has been determined to be 1.6 kb, of which no more than 1.2 kb codes for the integrase protein. A nonsense mutant defective in integration has been isolated by using a rapid screening procedure that identifies unstable plasmids.     

Low temperature and glucose enhanced T7 RNA polymerase-based plasmid stability for increasing expression of glucagon-like peptide-2 in Escherichia coli

The high activity of T7 RNA polymerase has made the T7 RNA polymerase-based expression system very powerful for high-level expression of recombinant protein. However, the overactivity of T7 RNA polymerase would also bring about negative effects on plasmid stability and protein production, especially when expressing a toxic protein. If the latter role is dominant, it is necessary to adopt some measures to attenuate the activity or the amount of T7 RNA polymerase in the cells. Apart from the stringent regulation by inserting some genes reducing the amount or the activity of T7 RNA polymerase into plasmids, optimizing the culture conditions would be another way. In this work, we have studied the effects of various culture conditions on the plasmid stability and the target protein yield including selective pressure, culture temperature, toxicity of the target protein and the catabolite repression caused by glucose. The results have indicated that adding antibiotic after induction has little effect in increasing plasmid stability, but inducing expression at low temperature and adding glucose to the medium improved the plasmid stability and the protein yield to a large extent.     

Stability of a host-vector system based on complementation of an essential gene in Escherichia coli.

Antibiotic selection is the most common selection system for plasmid-containing bacteria. This technique, nevertheless, can be a source of problems during the expression of heterologous genes in Escherichia coli. We have developed an alternative selection system based on the complementation of a chromosomal auxotrophic (dapD2) mutation by the corresponding wild type gene carried on a plasmid. We show that the system effectively selects for the presence of plasmid on solid and liquid medium. In addition, we have observed a loss of viability associated with high levels of gene expression and accumulation of a heterologous protein, but the selective power and improved intrinsic stability of the dap+ plasmid, compared to a beta-lactamase (bla) based vector, excludes overgrowth of the culture by plasmidless cells.     

In vivo transfer of chromosomal mutations onto multicopy plasmids by transduction with bacteriophage P1

A technique is presented by which chromosomal mutations may be efficiently transferred onto chimeric multicopy plasmids in vivo. The technique employs the transduction of plasmids using bacteriophage P1 as vector. The utility of this method was demonstrated by cloning a chromosomal ompR mutation of Escherichia coli K-12. The high-frequency transduction of the chimeric plasmid appeared to be dependent on its integration into the chromosome by homologous recombination. The results also suggest that the plasmid was transduced as part of the chromosome and resolved from its integrated state in the recipient cell, resulting in a high yield of mutant plasmid segregants.     

Application of the new ccdAB-type natural toxin-antitoxin module for stabilization of inheritance of expressive plasmid vectors based on the bacteriophage N15 replicon in Escherichia coli cells

Biopharmaceutical industry currently produces considerable quantity of novel recombinant preparations by way of overexpression in Escherichia coli cells, an inexpensive, efficient, time-proven, and practically feasible system of heterologous expression. Due to the instability of maintenance and inheritance of expression vectors in producer cells, the cells that have spontaneously lost the plasmid gain a significant selective advantage over the cells producing a heterologous protein and accumulate in the fermentor. For solution of this problem, it is proposed to develop a new generation of expression vectors with high stability of inheritance in the absence of external selective pressure, using a replicon of phage N15, which possesses its own system for active distribution of plasmid copies in the daughter cells, supplemented by a toxin-antitoxin genetic module preventing the loss of a plasmid. Two new addiction modules homologous to the known ccdAB and mazEF systems were isolated from natural enterobacterial populations and characterized. The testing showed more effective operation of the ccdAB module. The latter was a basis for construction of new expression vectors pN15E41 and pN15E61 demonstrating the high synergism of action of the plasmid segregation systems and the addiction module and directly applicable for biotechnological practice.     

Construction of <Emphasis Type="Italic">Streptomyces nogalater</Emphasis> Lv65 strains with enhanced nogalamicin biosynthesis using regulatory genes

Influence of cloned regulatory genes on nogalamycin biosynthesis by Streptomyces nogalater LV65 strain has been studied. Gene snorA from the S. nogalater genome was cloned in multicopy replicative plasmid pSOKA and integrative plasmid pR3A. Introduction of these plasmids into S. nogalater wild type cells resulted in enhanced nogalamicin biosynthesis. A similar effect was observed at heterologous expression of gene (p)ppGpp-synthetase gene relA cloned from Streptomyces coelicolor A3(2). Heterologous expression of genes absA2 from Streptomyces ghanaensis ATCC14672 and lndYR from genome Streptomyces globisporus 1912 decreased synthesis of antibiotic. The study results indicate the presence of homologs of these genes in chromosome of S. nogalater, their possible participation in regulation of nogalamicin biosynthesis, and provide us with a possibility for genetic design of the strains with higher synthesis of this antibiotic.     

Mitotic gene conversion of large DNA heterologies in Saccharomyces cerevisiae

Summary Gene conversion of large DNA heterologous fragments has been shown to take place efficiently in Saccharomyces cerevisiae. It has been found that a 2.6 kb LEU2 DNA fragment in a multicopy plasmid was replaced by a 3.1 kb PG11 chromosomal DNA fragment, when both fragments were flanked by homologous DNA regions. Gene conversion was asymmetric in a total of 481 recombinants analyzed. In contrast, truncated PG11 or LEU2 genes in multicopy plasmids, gave no recombinants that restored a complete plasmid copy of these genes in a total of 242 recombinants studied, confirming that a conversion tract is disrupted by a heterologous region. The asymmetry of the events detected suggest that gene conversion of large DNA heterologies involves a process whereby a gap first covers one heterologous fragment and then this is followed by new DNA synthesis using the other heterologous fragment as a template. Therefore, it is likely that large DNA heterologies are converted by a double-strand gap repair mechanism.     

Impact of plasmid architecture on stability and yEGFP3 reporter gene expression in a set of isomeric multicopy vectors in yeast

Multicopy episomal plasmids in yeast, used whenever elevated levels of foreign or homologous gene expression are necessary, are known to be less stable compared to the endogenous 2-μm plasmid they are based on, at least without selective pressure. Considering that rich medium favors growth rate and, simultaneously, is less expensive than selective medium, enhancing stability in non-selective medium is extremely desirable. In this study, we changed the architecture of a multicopy model expression plasmid, creating six isoforms (same size, same DNA content but different positions and orientations of the expression block) and studied mitotic stability, copy number, as well as reporter yEGFP3 expression between isoforms. With one isoform being significantly more stable than the others and another one exhibiting elevated plasmid copy numbers in rich medium, we show that consideration of the arrangement of the plasmid elements might be crucial for productivity employing Saccharomyces cerevisiae as a host. We strongly believe that the ideal architecture has to be assessed for each case and assembly strategy has to begin by evaluating the stability of the vector backbone before insertion of the desired gene. For the plasmid set studied, yEGFP3 reporter production depends more on mitotic stability than on elevated plasmid copy numbers in a small number of cells retaining the plasmid under non-selective conditions.

     

Identification of multicopy suppressors of the pcnB plasmid copy number defect in Escherichia coli

Plasmids containing a ColE1 origin of replication are widely used for cloning purposes in Escherichia coli. Among the host factors that affect the copy number of ColE1 plasmids is the E. coli protein poly(A) polymerase I (PAP I), which regulates the intracellular level of RNA I, a ColE1-encoded negative regulator of plasmid replication. In strains that lack PAP I, RNA I levels are elevated, resulting in reduced levels of ColE1 plasmids in the cell. PAP I is encoded by the gene pcnB. We devised a genetic approach, based on the identification of multicopy suppressor clones, to identify trans-acting factors that can help offset the ColE1 plasmid copy number defect in a pcnB (-) genetic background. Using this strategy, we identified suppressors that mapped to two regions of the E. coli chromosome. The suppressor activity of one of the chromosomal regions was localized to the rssB gene, a response regulator gene known to be involved in the turnover of the stationary-phase sigma factor, RpoS. The second suppressor maps to min 55.4 of the E. coli chromosome, and the factor responsible for the suppressor activity appears to be a novel RNA or protein.     

Multicopy integration and expression of heterologous genes in Methylobacterium extorquens ATCC 55366

High-level expression of chromosomally integrated genes in Methylobacterium extorquens ATCC 55366 was achieved under the control of the strong M. extorquens AM1 methanol dehydrogenase promoter (PmxaF) using the mini-Tn7 transposon system. Stable maintenance and expression of the integrated genes were obtained in the absence of antibiotic selective pressure. Furthermore, using this technology, a multicopy integration protocol for M. extorquens was also developed. Chromosomal integration of one to five copies of the gene encoding the green fluorescent protein (gfp) was achieved. The multicopy-based expression system permitted expression of a preset number of gene copies. A unique specific Tn7 integration locus in the chromosome of M. extorquens, known as the Tn7 attachment site (attTn7 site), was identified. This single attTn7 site was identified in an intergenic region between glmS, which encodes the essential enzyme glucosamine-6-phosphate synthetase, and dhaT, which encodes 1,3-propanediol dehydrogenase. The fact that the integration event is site specific and the fact that the attTn7 site is a noncoding region of the chromosome make the mini-Tn7 transposon system very useful for insertion of target genes and subsequent expression. In all transformants tested, expression and segregation of the transforming gene were stable without generation of secondary mutations in the host. In this paper, we describe single and multicopy chromosome integration and stable expression of heterologous genes (bgl [beta-galactosidase], est [esterase], and gfp [green fluorescent protein]) in M. extorquens.     

Molecular cloning of pheR in Escherichia coli K-12.

The regulator gene pheR, which in Escherichia coli controls the expression of pheA, the structural gene for chorismate mutase P-prephenate dehydratase, was cloned on to multicopy plasmids directly from the E. coli chromosome; this was achieved with the aid of the tetracycline resistance transposon, Tn10, that had been inserted very close to the pheR gene. Subsequently, pheR was subcloned on a 1.1-kilobase-pair fragment on the plasmid vector pBR322; its position on the plasmid was localized by the method of gamma delta-mediated transpositional inactivation. The pheR gene product was identified in maxicells and found to be a protein of subunit molecular weight 19,000, suggesting that the coding segment of the gene is about 500 nucleotide pairs long.     

Molecular cloning of plasmid ColIb-P9 genes responsible for its mutator function

The localization of plasmid ColIb-P9 muc genes mediating the plasmids protective and mutagenesis-increasing activity has been determined. The increase of muc genes dose by cloning them within the multicopy vector has been shown to repress the mutator function of the plasmid. No essential homology has been revealed between ColIb-P9 muc gene nucleotide sequences, pKM101 muc genes with a similar function, and umuDC chromosome genes. It has been shown that the synthesis of 38 KD protein is essential for the manifestation of the mutator function of the plasmid.